As it is well known, various proteases, such as trypsin and chymotrypsin, play their roles in the body, and they take important roles in vivo such as digestion, defense mechanism, blood coagulation and fibrinolysis and the like. On the other hand, it has been revealed by previous studies that some diseases and syndromes are caused by abnormal activities of these proteases. For example, autolysis of the pancreas by abnormally activated trypsin is regarded as a cause of the onset of pancreatitis. Certain proteases such as elastase and tissue kallikrein which are leaked from disordered tissues, leucocytes and the like, as well as plasma proteases such as plasmin and plasma kallikrein, are taking part of the development of tissue degradation. In the events of shock, by proteases leaked from damaged cells, blood circulation in important organs is decreased and progressive functional disorders occur in these organs. In addition, it is considered that excess activation of proteases involved in blood coagulation is a direct cause of disseminated intravascular coagulation syndrome (DIC) which is often induced by several kinds of shocks, severe infectious diseases and the like.
In consequence, protease inhibitors which inhibit activities of the aforementioned proteases have been used in the treatment of diseases such as pancreatitis, shock, DIC, organ disorder and the like.
The protease inhibitors so far used as pharmaceuticals are divided into two groups, namely chemically synthesized compounds and natural substances. Typical examples of the former group include nafamostat mesilate and gabexate mesilate while the latter group includes aprotinin and urinary trypsin inhibitor (to be referred to as "UTI" hereinafter). Of these inhibitors, aprotinin is a protein purified from bovine lung and UTI is another protein purified from human urine.
Various types of proteinous protease inhibitors exist in the body, such as .alpha.1 antitrypsin and .alpha.2 macroglobulin. These proteinous protein inhibitors are considered to have a function to maintain homeostasis by regulating activities of proteases in vivo. Thus, it is expected that these proteinous protein inhibitors, when used as pharmaceuticals, will not only simply inhibit protease activities but also improve a disordered state. For example, it has been reported that the aforementioned aprotinin has an activity to improve liver function (Nakamura, T. et al., Kanzo, vol. 28, pp. 1257-1258, 1987) and that a urinary glycoproteinous protease inhibitor stimulates growth of blood vessel endothelial cells (Mckeehan, W. L., The Journal of Biological Chemistry, vol. 261, pp. 5378-5383, 1986). Because of such possible additional biological activities which cannot be found in chemically synthesized protease inhibitors, usefulness of these proteinous protease inhibitors as pharmaceuticals is evident.
The following describes properties of UTI.
UTI is a proteinous protease inhibitor initially purified from healthy human urine in accordance by the method of Proksch et al. (Proksch, G. J. et al., J. Lab. Clin. Med., vol. 79, pp. 491-499, 1972), with the molecular weight of about 67,000 by gel filtration (Sumi H. et al., J. Physiol. Soc. Japan, vol. 39, pp 59-61, 1977).
Various types of urinary trypsin inhibitors with different molecular weights have been reported. A typical well-studied example among these inhibitors is a proteinous protease inhibitor having a molecular weight of 30,000 which has been purified from human urine by Hochstrasser et al. who also have determined primary structure of the inhibitor (Hochstrasser et al., Hoppe-Seyler's Z. Physiol. Chem., vol. 362, pp. 1351-1355, 1981). According to Hochstrasser et al., this inhibitor is a protein composed of 143 amino acids having two active domains. Based on the secondary structure of these active domains, this inhibitor can be regarded as a member of the Kunitz-type protease inhibitors which also include aprotinin. It is also known that this inhibitor coincides with the light chain of a plasma protease inhibitor, inter-.alpha.-trypsin inhibitor (to be referred to as "ITI" hereinafter) (Bourgunion, J. et al, Biochemical Biophysical Research Communications, vol. 131, pp. 1146-1153, 1985).
This inhibitor reported by Hochstrasser et al. is different from UTI in terms of their molecular weights, but their N-terminal sequence of 36 amino acids coincides with each other (Sumi, H. et al, Blood & Vessel, vol. 19, pp. 545-557, 1988). It has been suggested that the difference in molecular weights between these two urinary trypsin inhibitors may be due to difference in their sugar moiety contents (Shikimi, T., Pharma Medica, vol. 7, pp. 169-174, 1989).
Next, enzyme inhibition activities of UTI are described.
Based on the studies so far reported, it is evident that UTI shows the inhibition activities against many proteases such as trypsin, chymotrypsin, elastase, plasmin and the like (Ohnishi, H. et al., Folia Pharmacol. Japon, vol. 81, pp. 235-244, 1983). As described in the foregoing, however, there are no reports on its activity to inhibit plasma kallikrein. In addition, when the inventors of the present invention have conducted experiments on the inhibitory activities of UTI, it showed no plasma kallikrein-inhibiting activity. In the case of inhibition of an activated form of blood coagulation factor X (to be referred to as "FXa" hereinafter), it has been reported that a very high concentration of UTI slightly inhibited the activity of FXa (Fukutake, K., Folia Pharmacol, Japan, vol. 90, pp. 163-169, 1987) and that a very high concentration of UTI inhibited the FXa activity (Sakuragawa, N, et al., Saishin-igaku, vol. 42, pp. 820-830, 1987). According to experiments conducted by the present inventors, however, no FXa-inhibiting activity was found in UTI even at an extremely high concentration. Similar to the reports mentioned above, there are no reports on the activity of the 30,000 molecular weight protease inhibitor purified from human urine by Hochstrasser et al. to inhibit FXa or plasma kallikrein.
The C-terminal side domain of this inhibitor has been examined in detail in terms of its enzyme inhibition activities. Even in the case of this domain, there are no reports on its activity to inhibit FXa or plasma kallikrein, though it is known that this domain inhibits trypsin, chymotrypsin and plasmin (Albrecht, G. J. et al., Hoppe-Seyler's Z. Physiol. Chem., vol. 364, pp. 1689-1696, 1983).
Attempts have been made to endow UTI with an additional inhibitory spectrum. For instance, Nishimaki et al. have prepared fragments of UTI by chemically modifying it with cyanogen bromide and cleaving its methionyl bond, and examined enzyme inhibiting activities of the resulting mixture of polypeptide fragments (Japanese Patent Publication No. 3-59079). Nishimaki et al., however, could not endow the fragments obtained by this process with an activity to inhibit FXa or plasma kallikrein.
As described in the foregoing, proteinous protease inhibitors have superior advantages as pharmaceuticals compared to chemically synthesized inhibitors. However, the currently used proteinous protein inhibitors as pharmaceuticals, namely aprotinin and UTI, have the following disadvantages.
In the case of aprotinin, it inhibits certain proteases such as trypsin, plasmin, kallikrein and the like (Yoshida, K. et al., Nihon Rinsho, vol. 48, pp. 165-172, 1990). Being bovine originated protein, however, it has a big possibility of causing anaphylactic shock because it shows antigenicity when non-orally administered to human, thus resulting in a serious problem in the case of its use as a pharmaceutical. In addition to this disadvantage, since related diseases due to protease activation are generally accompanied by the stimulation of blood coagulation in many cases, it is desirable that a protease inhibitor to be used for therapeutic purpose should possess the activity to inhibit a certain protease such as FXa which takes a main role in the blood coagulation cascade, in addition to its activities to inhibit other proteases such as trypsin and the like. However, we know of no report concerning the activity of aprotinin to inhibit FXa.
On the contrary, UTI is more preferable than aprotinin as a drug to be non-orally administered to human, because this protein is a human protein and therefore it shows no antigenicity against human. However, since UTI is a proteinous substance which is purified from human urine, it is difficult to obtain enough quantity of human urine when UTI is manufactured in a large scale. It is also difficult to produce this substance in a large quantity by means of genetic engineering techniques, because it has a large molecular weight and also is a glycoprotein. In addition, similar to the case of aprotinin, UTI has neither a FXa-inhibiting activity nor an activity to inhibit plasma kallikrein which is concerned in severe inflammation. In consequence, these proteinous protein inhibitors currently used as pharmaceuticals have many problems to be solved. It is necessary therefore to overcome these problems involved in the prior art by finding a novel substance which can inhibit disease-related proteases, as well as a process for the production of such a substance, thereby rendering possible treatment of diseases in which proteases are acting as key factors.